Fig. 2.

Paracrine factors released from cultured astrocytes prevent intraneuronal oligomerization and interneuronal propagation of α-syn. (A and B) Paracrine effect of cultured astrocytes on the levels of α-syn aggregation. Media conditioned in astrocyte (ACM) or control Ctx-NSC (C-CM) cultures were collected and treated to α-syn–expressing mDA neuron-enriched cultures. Fourteen to 20 d after α-syn PFF treatment, α-syn aggregation was detected by immunoreactivity against pS129-αsyn and Thioflavin S staining. (C and D) Intracellular di-/oligomerization detected by α-syn BiFC system. (C) Schematic of the experimental procedures with BiFC system. BiFC+ inclusions (yellow puncta) are counted in D. (E and F) Interneuronal α-syn transmission. In the dual-chamber device, in which SH-SY5Y cells transduced with GFP-labeled A53T α-syn and those of nontransduced were plated in the upper and lower chambers, respectively (1 × 10⁴ cells in each chamber, schematized in E), neuron-to-neuron α-syn transmission was estimated by counting A53T-αsyn-GFP puncta intensity (F). GFP fluorescence intensity was measured using ImageJ software and presented as mean fluorescence intensity (MFI). Significant differences from the α-syn–treated (+) and CM-untreated (−) control at ^##P < 0.01 and ^###P < 0.001 and between the groups indicated at *P < 0.05, **P < 0.01, and ***P < 0.001, n = 3 to 5 cultures (B–D), 13 to 89 cells (F), one-way ANOVA. ns, no significance. (Scale bars: 25 μm.)